Purification of guinea pig C9 will be improved by the use of ion- exchange chromatography isoelectric focusing, and isotachophoresis. A monospecific antibody will be prepared to pure C9. Steps will be devised for purification of bovine serum C9. After precipitation of C9 in bovine serum with ammonium sulfate, the fraction containing C9 will be chromatographed on hydroxyapatite, DEAE-cellulose, CM-cellulose and Sephadex G-200. A final step of isoelectric focusing and/or isotachophoresis may yield a homogeneous product. The procedure will be scaled up in ca. 5-fold increments until 5-10 L can be processed at once. The mechanism by which C9 causes membrane damage will be investigated. Previous results of effect of temperature on kinetics of the reaction will be confirmed. The OD191 method of detection of protein in the microgram range will be adapted to accurately measure protein concentration of purified guinea pig and bovine C9. This data, along with previous molecular weight estimates by sucrose density sedimentation and hemolytic activity titration, will be used to determine the efficiency of the reaction. Binding of C9 to the membrane will be measured by uptake of radioiodinated C9 by immune complexes possessing C8 sites, agglutination of EAC142356789 before and after lysis by anti-C9 and removal of C9 neutralizing activity from anti-C9 by immune complexes possessing C8 sites. Liberation of molecular fragments of C9 as a result of reaction will be examined by analysis of reaction mixture supernatant fluids immunoelectrophoretically, by molecular seiving and ion-exchange chromatography using the OD191 protein detection method, or a neutralization competition type assay. Lastly, studies of synthesis of C9 and albumin by a stable rat hepatoma tissue culture cell line will be continued. Effects of anti-inflammatory drugs on synthetic capabilities will be evaluated and attempts will be made to achieve incorporation of C14-amino acids into the C9 molecule. Other tissue culture cell lines will be examined for C9 as well as other complement components.